Light sheet fluorescence microscopy (LSFM) is an imaging method that uses one or more illumination objectives to excite fluorophores in a specimen with a thin sheet of light. Emitted light from the sample is then visualized by a separate imaging objective that is perpendicular to the illuminating lightsheet.
Light sheet microscopy (LSFM) has several advantages over confocal microscopy (LSCM):
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Excellent sample penetration- In conventional confocal microscopy, excitation illumination must pass from the objective through the sample to the plane of focus, then emitted light must return through the sample and into the objective via the same light path. The compounded scattering of both excitatory and emitted light limits sensitivity and resolution in thick samples. In LSFM, the excitation light sheet comes from dedicated illumination objectives and emitted light from the sample is detected by a separate imaging objective. This reduces light scattering and improves imaging in thick specimens.
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High speed- In confocal microscopy, the excitation laser scans across the sample field and emitted light is captured by a photo-multiplier tube, constructing an image pixel by pixel. In LSFM, The entire focal plane is illuminated at once, allowing an image to be collected using a high speed camera.
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Minimal phototoxicity and photobleaching- Because excitatory light is passing through the entire depth of the sample in confocal microscopy, there can be significant bleaching of the sample and a high risk for phototoxicity in live specimens. Because only a thin sheet of excitatory light is used in LSFM and because imaging with a camera is rapid, this method dramatically reduces photobleaching and phototoxicity.
These properties make LSFM ideal for many biological imaging applications, including:
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Fluorescence imaging of thick samples
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Long term imaging of live samples
For more information about LSFM, visit MicroscopyU.
(Image courtesy of Carl Zeiss AG)
